Toxicity of smokeless tobacco in human oral epithelium with emphasis on carcinogen metabolism and regulation of programmed cell death
Author: Vondracek, Martin
Date: 2002-10-31
Location: Rockefellersalen, Nobels väg 11
Time: 9.15
Department: Institutet för miljömedicin (IMM) / Institute of Enviromental Medicine
View/ Open:
thesis.pdf (795.7Kb)
Abstract
The oral mucosa is globally a common site for cancer development. Primary
risk factors include tobacco smoking and alcohol consumption whereas the
contribution from usage of smokeless tobacco remains debated. The
susceptibility of the human oral epithelium to carcinogens in tobacco
likely depends on the presence of biotransformation enzymes, capable of
metabolically activating or detoxifying these agents as well opposing
influences from oxidative stress. Induction of programmed cell death
(PCD), including function of tumor suppressor p53, may also modulate
smokeless tobacco toxicity. On this basis, the purpose of this study was
to investigate the expression of biotransformation enzymes as well as the
roles of PCD and p53 in smokeless tobacco toxicity in oral epithelium.
Various qualitative and quantitative analyses of oral tissue specimens
and normal, immortalized and malignant oral keratinocytes indicated
presence of multiple biotransformation enzymes. Several cytochrome P450
(CYP) transcripts were demonstrated including ]AL 1A2, 2C, 2D6, 2E1,
3A4/7 and 3A5. 'typical CYP substrates, including ethoxyresorutin,
methoxyresorufin and chlorzoxazone, were delectably oxidized in vitro and
metabolism of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)1-
(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1) resulted in
covalently bound adducts. Moreover, normal keratinocytes and SV40T
antigen-immortalized keratinocytes (SVpgC2a) were shown to express
enzymes catalyzing conjugation reactions and detoxification of reactive
oxygen. Notably, SVpgC2a showed higher expression levels than normal
keratinocytes of some enzymes, e.g. CYP1B1, By RT-PCR, the CYPs where
generally shown to be expressed at levels <50 molecules, the conjugation
enzymes at levels between 50-1000 molecules and the enzymes involved in
detoxification of reactive oxygen at levels >1000 molecules, using 106
molecules of beta-actin as reference. Microarray analysis confirmed
expression of these enzymes at levels >300 molecules per 1 06 molecules
of beta-actin. The results indicated presence of several
biotransformation enzymes in oral buccal mucosa in vivo and in vitro,
indicating the usefulness of oral keratinocyte cell lines for studies of
both single agents and complex mixtures in human oral epithelium.
Studies of smokeless tobacco toxicity involved cultured oral keratinocyte
cell lines and oral tissue specimens obtained from healthy controls,
snuff users (SDL) and patients diagnosed for lichen planus (OLP).
Assessments of net growth rates. apoptosis, necrosis and terminal
differentiation in vitro showed that aqueous smokeless tobacco extract
prepared from "Ettans snus" (STE) primarily caused necrotic death without
substantial involvement of PCD. Carcinoma cells (SqCC/Y1) were more
resistant to necrosis from STE as compared to normal cells. Extract
prepared from "Kentucky standard reference tobacco" caused similar
toxicity as STE. The latter extract induced increases in p53 content that
did not associate to increased apoptosis, whereas in contrast. the DNA
damaging agent mitomycin C (MMC) increased both p53-content and
apoptosis. STE and nicotine separately. significantly inhibited apoptosis
induced by various regimens. Slight increases in bcl-2 transcripts in
STE-exposed keratinocytes indicated the involvement of this gene.
Analysis of Jurkat cells implied that reactive smokeless tobacco
chemicals might also block apoptosis by inhibiting caspase activity. Oral
tissue analysis agreed with the concept that smokeless tobacco may
inhibit apoptosis, i.e. increased mitosis in SDL (relative to normal
controls) was not associated with increased apoptosis, whereas OLP
exhibited increases in both mitosis and apoptosis. Finally, expression of
the p53 and Bcl-2 proteins was noted in SDL whereas OLP expressed p53 but
not bcl-2.
In summary, the analysis of the expression of biotransformation enzymes
and smokeless tobacco toxicity generally demonstrated similar results in
tissue and cultured cell lines implying the usefulness of cell culture
technology in the investigation of mechanisms underlying carcinogenesis
and other oral disease processes. Thus. keratinocytes actively expressing
multiple biotransformation enzymes were susceptible to smokeless tobacco
toxicity. The toxicity mechanism of smokeless tobacco likely involves
metabolism of carcinogenic agents, including N-nitrosamines. and
inhibition of p53-mediated apoptosis. Thus, this study suggests several
mechanisms whereby smokeless tobacco usage may contribute to adverse
health effects including those associated with cancer development in the
oral epithelium.
List of papers:
I. Vondracek M, Xi Z, Larsson P, Baker V, Mace K, Pfeifer A, Tjalve H, Donato MT, Gomez-Lechon MJ, Grafstrom RC (2001). "Cytochrome P450 expression and related metabolism in human buccal mucosa. " Carcinogenesis 22(3): 481-8
Pubmed
II. Crawford EL, Peters GJ, Noordhuis P, Rots MG, Vondracek M, Grafstrom RC, Lieuallen K, Lennon G, Zahorchak RJ, Georgeson MJ, Wali A, Lechner JF, Fan PS, Kahaleh MB, Khuder SA, Warner KA, Weaver DA, Willey JC (2001). "Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR. " Mol Diagn 6(4): 217-25
Pubmed
III. Vondracek M, Weaver DA, Sarang Z, Hedberg JJ, Willey JC, Warngard L, Grafstrom RC (2002). "Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes. " Int J Cancer 99(6): 776-82
Pubmed
IV. Vondracek M, Hansson A, Zheng X, Sand L, Noren U, Hirsch JM, Lindholm J, Grafstrom RC (2002). "Smokeless tobacco toxicity and influences on programmed cell death in human oral mucosa in vivo and in vitro." (Manuscript)
V. Vondracek M, Zheng X, Noren U, Elfwing A, Grafstrom RC (2002). "Influences of smokeless tobacco extract on growth and apoptosis in cultured human normal and malignant buccal keratinocytes." (Manuscript)
I. Vondracek M, Xi Z, Larsson P, Baker V, Mace K, Pfeifer A, Tjalve H, Donato MT, Gomez-Lechon MJ, Grafstrom RC (2001). "Cytochrome P450 expression and related metabolism in human buccal mucosa. " Carcinogenesis 22(3): 481-8
Pubmed
II. Crawford EL, Peters GJ, Noordhuis P, Rots MG, Vondracek M, Grafstrom RC, Lieuallen K, Lennon G, Zahorchak RJ, Georgeson MJ, Wali A, Lechner JF, Fan PS, Kahaleh MB, Khuder SA, Warner KA, Weaver DA, Willey JC (2001). "Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR. " Mol Diagn 6(4): 217-25
Pubmed
III. Vondracek M, Weaver DA, Sarang Z, Hedberg JJ, Willey JC, Warngard L, Grafstrom RC (2002). "Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes. " Int J Cancer 99(6): 776-82
Pubmed
IV. Vondracek M, Hansson A, Zheng X, Sand L, Noren U, Hirsch JM, Lindholm J, Grafstrom RC (2002). "Smokeless tobacco toxicity and influences on programmed cell death in human oral mucosa in vivo and in vitro." (Manuscript)
V. Vondracek M, Zheng X, Noren U, Elfwing A, Grafstrom RC (2002). "Influences of smokeless tobacco extract on growth and apoptosis in cultured human normal and malignant buccal keratinocytes." (Manuscript)
Issue date: 2002-10-10
Rights:
Publication year: 2002
ISBN: 91-7349-335-x
Statistics
Total Visits
Views | |
---|---|
Toxicity ...(legacy) | 801 |
Toxicity ... | 113 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Toxicity ... | 0 | 2 | 1 | 2 | 1 | 2 | 1 |
File Visits
Views | |
---|---|
thesis.pdf | 2012 |
thesis.pdf(legacy) | 621 |
thesis.pdf.txt(legacy) | 2 |
Top country views
Views | |
---|---|
United States | 377 |
China | 74 |
Germany | 48 |
Sweden | 41 |
India | 23 |
South Korea | 20 |
Denmark | 15 |
France | 12 |
Malaysia | 10 |
United Kingdom | 9 |
Top cities views
Views | |
---|---|
Beijing | 33 |
Sunnyvale | 32 |
Romeo | 20 |
Kiez | 18 |
Seoul | 14 |
Ballerup | 13 |
Penang | 10 |
Ashburn | 7 |
Dublin | 7 |
London | 6 |